1. Field of the Invention
This invention concerns an improved process for the production of glyoxylic acid by the glycolate oxidase catalyzed oxidation of glycolic acid. Although the enzyme catalyzed reaction of glycolic acid with oxygen has been known for many years, the previously described processes have not proved commercially advantageous for several reasons. The most important of these is that previous reactions have been carried out at high dilutions of glycolic acid, typically concentrations of 40 mM or less. Selectivity for glyoxylic acid and yields of the glyoxylic acid product have usually been low. A disadvantage of the use of very dilute starting glycolic acid concentrations is the necessity for large and expensive reaction vessels to achieve high production rates. Also, since glyoxylic acid is usually sold as a 50% aqueous solution (Ullmans), concentrating the dilute glyoxylic acid produced using dilute starting reagents is costly. Further, if such concentration were done by evaporation or by reverse osmosis, any non-volatile by-products such as oxalic and formic acids and/or their salts and unreacted glycolic acid would remain in solution as impurities. Finally, it would be advantageous if the relatively expensive enzymes used in the reaction could be used more efficiently or effectively recycled and if no environmentally detrimental wastes were produced.
The present invention provides a commercially practical method for the production of glyoxylic acid by the glycolate oxidase catalyzed oxidation of glycolic acid through the increase of starting substrate concentrations and through the use of selected yield improving additives.
2. Background Art
The present invention is a process for the production of glyoxylic acid by the oxidation of glycolic acid by oxygen, using an enzyme, glycolate oxidase, as a catalyst for the reaction.
N. E. Tolbert et al., J. Biol. Chem., Vol. 181, 905-914 (1949) reported that an enzyme extracted from tobacco leaves catalyzed the oxidation of glycolic acid to formic acid and CO.sub.2 via the intermediate glyoxylic acid. They further found that certain compounds such as ethylenediamine blocked the oxidation of the intermediate glyoxylic acid to other products. The oxidations were carried out at a pH of about 8, using glycolic acid concentrations of about 3-40 mM (millimolar), except for one experiment (p. 907), very poorly described, where the initial concentration of glycolic acid was somewhere between 132 and 196 mM. The only details given about this experiment are the approximate glycolic acid concentration, the fact that the oxidation was not run to completion, and that some amount of the 2,4-dinitrophenylhydrazone of glyoxylic acid was isolated. In particular, no details are given as to yields and the duration of the reaction. The optimum pH for the glycolate oxidation was reported to be 8.9. Oxalic acid (100 mM) was reported to inhibit the catalytic action of the glycolate oxidase.
I. Zelitch and S. Ochoa, J. Biol. Chem., Vol. 201, 707-718 (1953) reported that the formation of formic acid and CO.sub.2 in the glycolate oxidase catalyzed oxidation of glycolic acid resulted from the nonenzymatic reaction of H.sub.2 O.sub.2 with glyoxylic acid, these being the primary products of the enzyme catalyzed oxidation of glycolic acid. Thus, they observed that addition of catalase, an enzyme that catalyzes the decomposition of H.sub.2 O.sub.2, greatly improved the yields of glyoxylic acid by suppressing the formation of formic acid and CO.sub.2. The glycolate oxidase they used was isolated from spinach leaves It was used at a pH of about 8, with an initial glycolic acid concentration of 10 mM. They also found that addition of FMN (flavin mononucleotide) greatly increased the efficiency of the glycolate oxidase.
J. C. Robinson et al., J. Biol. Chem., Vol. 237, 2001-2009 (1962) also found that catalase increases the yield of glyoxylic acid from glycolic acid. They apparently used a ratio of about 80:1 of catalase:glycolate oxidase. They also concluded that the catalase was decomposing hydrogen peroxide produced in the glycolate oxidase catalyzed reaction of glycolic acid with oxygen (in their paper, glycolate oxidase is referred to as "short chain L-alpha-hdyroxy acid oxidase"). They found that FMN was helpful in maintaining glycolate oxidase activity. They also determined that the maximum rate of oxidation of glycolic acid catalyzed by glycolate oxidase occurs at a concentration of glycolic acid (substrate) of 3.3 mM and that, "The reaction was found to be inhibited, by: . . . (e) high concentrations of these substrates, glycolate, and . . .".
K. E. Richardson and N. E. Tolbert, J. Biol. Chem., Vol. 236, 1280-1284 (1961) showed that buffers containing tris(hydroxymethyl)aminomethane inhibited the formation of oxalic acid in the glycolate oxidase catalyzed oxidation of glycolic acid. They too ran their reaction at a pH of about 8 and found that FMN increased glycolate oxidase efficiency. The maximum glycolic acid concentration they used was 20 mM.
C. O. Clagett, N. E. Tolbert and R. H. Burris, J. Biol. Chem., Vol. 178, 977-987 (1949) discovered that the optimum pH for the glycolate oxidase catalyzed oxidation of glycolic acid with oxygen was about 7.8-8.6, and the optimum temperature was 35.degree.-40.degree. C. Their maximum substrate (glycolic acid) concentration was about 20 mM.
There are numerous other references to the oxidation of glycolic acid catalyzed by glycolic acid oxidase, for example:
Isolation of the enzyme (usually includes an assay method):
I. Zelitch in Methods of Enzymology, Vol. 1, Academic Press, New York, 1955, p. 528-532, from spinach and tobacco leaves. PA0 M. Nishimura et al., Arch. Biochem. Biophys., Vol. 222, 397-402 (1983), from pumpkin cotyledons. PA0 H. Asker and D. Davies, Biochim. Biophys. Acta, Vol. 761, 103-108 (1983), from rat liver. PA0 M. J. Emes and K. H. Erismann, Int. J. Biochem., Vol. 16, 1373-1378 (1984), from Lemna Minor L. PA0 E. Cederlund et al., Eur. J. Biochem., Vol. 173, 523-530 (1988). PA0 Y. Lindquist and C. Branden, J. Biol. Chem., Vol. 264, 3624-3628 (1989).
Structure of the enzyme:
In all of the above references, and all others that have been studied [with the one exception noted above in the discussion of N. E. Tolbert et al., J. Biol. Chem., Vol. 181, 905-914 (1949)], the maximum initial concentration of glycolic acid that has been used is about 40 mM, the pH has usually been about 8-9, FMN is sometimes added, an amine is sometimes added, and catalase is sometimes added. Other additives to improve the yield of glyoxylic acid have also been mentioned.
Numerous ordinary chemical (nonenzymatic) methods for the industrial synthesis of glyoxylic acid have been proposed, see for example U.S. Pat. Nos. 3,281,460, 4,146,731 and 4,235,684, as well as Ullmanns Encyklopadie der technischen Chemie, 4th Ed., Vol. 12, Verlag Chemie, Weinheim, 1976, p. 381 (herein Ullmanns). Some of these processes produce environmentally injurious products. None of these contemplate the oxidation of glycolic acid to glyoxylic acid.
Even though glycolic acid is an article of commerce, to Applicant's knowledge no one has previously contemplated using the glycolate oxidase catalyzed oxidation of glycolic acid for the production of glyoxylic acid. It is speculated that this may be due to the unfamiliarity of chemists and chemical engineers with enzyme reactions (biochemistry), the lack of recognition by biochemists that such a process was desirable, the relatively low yields or conversions reported in most of the literature, reported substrate inhibition and/or the low concentrations of substrate previously used.